Laminins are large heterotrimeric glycoproteins of the extracellular matrix. Each laminin heterotrimer is composed of an xcex1, a xcex2, and a xcex3 chain, chosen from a number of possible homologues of each chain. Currently, eleven laminin chains have been identified: five xcex1 chains, three xcex2 chains, and three xcex3 chains (Timp1 (1996) Curr Opin Cell Biol 8: 618-624).
The invention is based, in part, on the discovery of a novel member of the laminin family, laminin 15. Accordingly, the invention features a purified or isolated preparation, a recombinant preparation, or a composition of laminin 15, which includes laminin chains xcex15, xcex22, and xcex33. In a preferred embodiment, the laminin 15 is a trimer of an xcex15, xcex22, and xcex33 chain. In a preferred embodiment the laminin 15 is human laminin 15.
In a preferred embodiment the xcex15 chain has a molecular weight of 380 kD, or 330 kD, the xcex22 chain has a molecular weight of 190 kD or 170 kD, the xcex33 chain has a molecular weight of 220 kD, 200 kD or 170 kD.
In another preferred embodiment, the xcex15 chain is reactive with or specifically binds an xcex15-specific antibody, e.g., the mouse monoclonal antibody 4C7 (Engvall et al. (1986) J Cell Biol 103:2457-2465), or an antibody of the same laminin chain-specificity, e.g., one which can compete for the 4C7 epitope. In another preferred embodiment, the xcex22 chain is reactive with or specifically binds a xcex22 specific antibody, e.g., a guinea pig polyclonal GP1 (Sanes et al. (1990) J Cell Biol 111:1685-1699), mouse monoclonal C4 (Sanes et al. (1983) Cold Spring Harb Symp Quant Biol 48: 667-678), or an antibody of the same laminin chain-specificity, e.g., one which can compete for the GP1 or C4 epitope. In another preferred embodiment, the xcex33 chain is reactive with or specifically binds xcex33 specific a antibody, e.g., the rabbit antibody R16 or the rabbit antibody R21 (Koch et al. (1999) J Cell Biol 145: 605-618), or an antibody of the same laminin chain specificity, e.g., one which competes for the R16 or R21 binding site.
In yet another preferred embodiment, the xcex15 chain has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In a preferred embodiment, the xcex15 chain is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 98%, 99% homologous to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In a preferred embodiment, the xcex15 chain differs from the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, by at least one, but less than 5, 10, 15 amino acid residues, e.g., by at least one, but less than 5, 10, 15 non-essential amino acid residues. Preferably, the xcex15 chain retains the ability to form a heterotrimer with the xcex22 chain and the xcex33 chain.
In another preferred embodiment, the xcex22 chain has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In a preferred embodiment, the xcex22 chain is at least 60%, 65%, 70%m, 75%, 80%, 85%, 90%, 95%, 98%, 99% homologous to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In a preferred embodiment, the xcex22 chain differs from the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8, by at least one, but less than 5, 10, 15 amino acid residues, e.g., by at least one, but less than 5, 10, 15 non-essential amino acid residues. Preferably, the xcex22 chain retains the ability to form a heterotrimer with the xcex15 chain and the xcex33 chain.
In another preferred embodiment, the xcex33 chain has the amino acid sequence of SEQ ID NO: 10. In a preferred embodiment, the xcex33 chain is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% 99% homologous to the amino acid sequence of SEQ ID NO: 10. In a preferred embodiment, the xcex33 chain differs from the amino acid sequence of SEQ ID NO: 10, by at least one, but less than 5, 10, 15 amino acid residues, e.g., by at least one, but less than 5, 10, 15 non-essential amino acid residues. Preferably, the xcex33 chain retains the ability to form a heterotrimer with the xcex15 chain and the xcex22 chain.
In another aspect, the invention features, a purified or isolated preparation, a recombinant preparation, or composition of laminin 15, which includes laminin chains xcex15, xcex22, xcex33. In a preferred embodiment, the laminin 15 is a trimer of an xcex15, xcex22, and xcex33 chain. In a preferred embodiment, the laminin 15 is human laminin 15.
The laminin chains of any laminin as disclosed herein can be the initial translation product or a degradation product, e.g., a naturally occurring degradation product of a laminin chain.
In another aspect, the invention features an isolated nucleic acid, e.g., DNA, RNA or cDNA encoding laminin 15, i.e., which encodes xcex15, xcex22, or xcex33. The isolated nucleic acid can be a combination of nucleic acids each encoding one or more laminin 15 chains or a single nucleic acid, e.g., if in a vector, one or more of the chains can be in one vector or each chain can be in a separate vector. The xcex15 can be, e.g., any xcex15 chain described herein. In a preferred embodiment, the nucleic acid encoding the xcex15 chain has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In a preferred embodiment, the nucleic acid encoding the xcex15 chain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology, or has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. In another preferred embodiment, the nucleic acid encoding the xcex15 chain hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. The xcex22 chain can be, e.g., any xcex22 chain described herein. In a preferred embodiment, the nucleic acid encoding the xcex22 chain has the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7. In a preferred embodiment, the nucleic acid encoding the xcex22 chain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology, or has the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7. In another preferred embodiment, the nucleic acid encoding the 132 chain hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7. The xcex33 chain can be, e.g., any xcex33 chain described herein. In a preferred embodiment, the nucleic acid encoding the xcex33 chain has the nucleotide sequence of SEQ ID NO: 9. In a preferred embodiment, the nucleic acid encoding the xcex33 chain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% homology, or has the nucleotide sequence of SEQ ID NO: 9. In another preferred embodiment, the nucleic acid encoding the xcex33 chain hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence of SEQ ID NO: 9.
In a preferred embodiment, the isolated nucleic acid can be expressed in one or more vectors, e.g., an expression vector or expressed directly in a cell. A vector (or vectors) containing a sequence corresponding to the sequence of the isolated nucleic acid can express the isolated nucleic acid in a suitable cell or a suitable in vitro environment.
In another aspect, the invention features producing laminin 15 from a cell transfected with nucleic acid encoding a laminin 15, e.g., a laminin 15 described herein.
In another aspect, the invention features producing laminin 15 from a cell transfected with nucleic acid which encodes one or more of an xcex15 chain, a xcex22 chain and/or a xcex33 chain, e.g., a nucleic acid described herein.
In another aspect, the invention features a recombinant laminin 15 which can be produced, e.g., by expressing the laminin chains of laminin 15 in a suitable cell host and under a condition suitable for the laminin chains to form laminin 15.
In a preferred embodiment, the laminin 15 differs from a naturally occurring laminin 15 by at least 1, but less than 5, 10, or 15 amino acid residues. In another embodiment, one, two, or each laminin chain of a laminin, differs from its naturally occurring counterpart by at least 1, but less than 5, 10, or 15 amino acid residues.
The invention provides a method for treating a disorder associated with abnormal functions of synapses, e.g., insufficient stability, viability, formation, and/or defective organization of synapses. The method comprises administering to a subject an effective amount of: laminin 15, laminin 14, or a combination thereof.
The invention further provides a method for modulating retinal development, e.g., in the subretinal space, in the interphotoreceptor matrix, and/or in the outer plexiform layer. The method comprises administering to a subject an effective amount of: laminin 15, laminin 14, or a combination thereof.
The invention provides a method for treating a disorder associated with: insufficient neural cell growth, healing and regeneration, e.g., axon outgrowth; a disorder associated with abnormal subretinal space or interphotoreceptor matrix (IPM) such as inadequate stability of IPM; a disorder associated with retina contact, continuity, and/or adhesion; a disorder associated with abnormal and/or insufficient formation of synapses; a disorder associated with viability of a neural cell, e.g., photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body, and/or synapses. The method comprises administering to a subject an effective amount of laminin 15, laminin 14, or a combination thereof.
Another feature of the present invention provides a method of treating a disorder associated with retinal abnormality, e.g., rod dystrophy, rod-cone dystrophy, macular degeneration, retinitis pigmentosa, or retinal detachment. The method includes administering to a subject an effective amount of: laminin 15, laminin 14, or a combination thereof
Another feature of the present invention provides a method of inducing neural cell growth and/or regeneration, e.g., axon outgrowth. The method includes administering to a subject an effective amount of laminin 15, laminin 14, or a combination thereof. In a preferred embodiment, the method can be used to induce neural cell growth or regeneration in the central nervous system (CNS) and/or the peripheral nervous system (PNS).
In a preferred embodiment, the method includes administering to a wound an effective amount of: laminin 15, laminin 14, or a combination thereof.
Still another feature of the invention provides a method of promoting a condition, e.g., promoting retina inter-photoreceptor matrix stability; promoting the production, stability, and/or development of a retina photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body, and/or synapses; promoting retinal contact, continuity, and/or adhesion; promoting the stability of synapses; and/or promoting the formation of synapses. The method includes administering an effective amount of: laminin 15, laminin 14, or a combination thereof.
Another feature of the invention provides a method for preparing an implant. For example, a method of preparing an implantable tip, an implantable catheter, a retinal implant, a timed releasing device, a neural cell growth guide, an artificial tissue, an implant of the central nervous system, or an implant of the peripheral nervous system. The method includes contacting, e.g., coating or incubating, the implant with laminin 15. In a preferred embodiment, laminin 15, laminin 14, or combinations thereof, can be used for treatment of a damaged eye, e.g., to increase photosentivity in an eye, e.g., by implanting a tip coated with laminin 15, laminin 14, or a combination thereof, into the eye.
In a preferred embodiment, the implant is a subretinal implant, e.g., subretinal microphotodiodes, a visual prosthesis, e.g., a photoreceptive prosthesis (e.g., as reviewed in Peachey, J Rehabil Res Dev (1999) 36(4):371-6), an implant for photoreceptor replacement, a phototransistor, or a subretinally implanted microphotodiode array (MPDA) implant. Such implants are described in Zrenner et al. (1997) Ophthalmic Res 29(5):269-80; Zrenner et al. (1999) Vision Res 39(15):2555-67, or in the abstract entitled xe2x80x9cCan Subretinal Microphotodiodes Successfully Replace Degenerated hotoreceptors?xe2x80x9d submitted by E. Zrenner et al. at the Vision Research Conference held on May 9, 1998. An example of a corneal keratoprosthesis (the Aachen-Keratoprosthesis) is described in Kompa et al. (2000) Int J Artif Organs 23(5):345-8.
A method of evaluating a compound for the ability to interact with, e.g., bind, a subject laminin 15 is provided. The method includes: contacting the compound with the subject laminin 15; and evaluating ability of the compound to interact with, e.g., to bind or form a complex with the subject laminin 15. This method can be performed in vitro, e.g., in a cell free system, or in vivo. This method can be used to identify naturally occurring molecules which interact with subject laminin 15. It can also be used to find natural or synthetic inhibitors of subject laminin 15. Screening methods are discussed in more detail below.
In one embodiment, an assay is a cell-based assay in which a cell which expresses laminin 15 or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate laminin 15 activity is determined.
The ability of the test compound to modulate laminin 15 binding to a compound, e.g., a laminin 15 substrate, or to bind to laminin 15 can also be evaluated.
Soluble and/or membrane-bound forms of isolated proteins (e.g., laminin 15 or biologically active portions thereof can be used in the cell-free assays of the invention. When membrane-bound forms of the protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton(copyright) X-100, Triton(copyright) X-114, Thesit(copyright), Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N, N-dimethyl-3-ammonio-1-propane sulfonate.
Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
In a preferred embodiment, the assay includes contacting laminin 15 or biologically active portion thereof with a known compound which binds laminin 15 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with laminin 15, wherein determining the ability of the test compound to interact with laminin 15 includes determining the ability of the test compound to preferentially bind to laminin 15 or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.
In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder, e.g., a disorder described herein.
In a preferred embodiment, the disorder is related to a lesion in or the misexpression of a gene which encodes one or more of a laminin 15 chain, e.g., one or more of an xcex15 chain, the xcex22 chain, and/or xcex33 chain.
Such disorders include, e.g., a disorder associated with the misexpression of a laminin 15 chain, a disorder associated with the central nervous system and/or the peripheral nervous system, a retinal disorder.
The method includes one or more of the following:
detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5xe2x80x2 control region;
detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene;
detecting, in a tissue of the subject, the misexpression of one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;
detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of laminin 15.
In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.
For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: or naturally occurring mutants thereof or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.
In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of laminin 15.
Methods of the invention can be used prenatally or to determine if a subject""s offspring will be at risk for a disorder.
In preferred embodiments the method includes determining the structure of one or more of a laminin 15 chain gene, e.g., one or more of an xcex15 chain, the xcex22 chain, or xcex33 chain gene, an abnormal structure being indicative of risk for the disorder.
In preferred embodiments the method includes contacting a sample from the subject with an antibody to one or more of the xcex15 chain, the xcex22 chain, or xcex33 chain, or a nucleic acid, which hybridizes specifically with the gene.
In another aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted expression of one or more of a laminin 15 chain or a laminin 15 activity, by administering to the subject laminin 15 or an agent which modulates expression of one or more laminin 15 chain or at least one laminin 15 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted laminin 15 activity or expression of one or more laminin 15 chain can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the laminin 15 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of laminin 15 aberrance, for example, a laminin 15 agonist or laminin 15 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
It is possible that some laminin 15 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.
The term xe2x80x9ceffective amountxe2x80x9d means the amount that is sufficient to reduce or alleviate at least one adverse effect or symptom of a disorder and/or to induce or enhance at least one biological activity of laminin 15. A biological activity of laminin 15 includes one or more of the ability to: 1) modulate retinal development, e.g., in the subretinal space, the interphotoreceptor matrix, the outer plexiform layer; 2) modulate, e.g., promote, neural cell growth and regeneration, e.g., axonal outgrowth; 3) modulate, e.g., promote, adhesion between cells and/or extracellular matrix, e.g., retinal contact; 4) modulate, e.g., promote, synaptic formation; 5) modulate, e.g., promote, viability of a neural cell, e.g., a neural retinal cell, e.g., a photoreceptor or an element thereof, e.g., outer segment, inner segment, cell body or synapses; 6) interact, e.g., form a complex, with a dystrophin and/or a P-dystroglycan. An effective amount can be determined by one skilled in the art, e.g., based on the disease stage, age, sex, and weight of the to be treated subject and the condition of the treatment. As a reference, the amount administered can be at a concentration of at least from about 0.1 to 500 pg/ml, from about 1 to 200 g/ml, from about 10 to 150 g/ml, or from about 10 to 100 g/ml.
The term xe2x80x9cisolated or purified nucleic acid moleculexe2x80x9d includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term xe2x80x9cisolatedxe2x80x9d includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an xe2x80x9cisolatedxe2x80x9d nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5xe2x80x2 and/or 3xe2x80x2 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of 5xe2x80x2 and/or 3xe2x80x2 nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an xe2x80x9cisolatedxe2x80x9d nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
As used herein, the term xe2x80x9chybridizes under stringent conditionsxe2x80x9d describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley andSons, N. Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. A preferred, example of stringent hybridization conditions are hybridization in 6xc3x97 sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97 SSC, 0.1% SDS at 50xc2x0 C. Another example of stringent hybridization conditions are hybridization in 6xc3x97 sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97 SSC, 0.1% SDS at 55xc2x0 C. A further example of stringent hybridization conditions are hybridization in 6xc3x97 sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97 SSC, 0.1% SDS at 60xc2x0 C. Preferably, stringent hybridization conditions are hybridization in 6xc3x97 sodium chloride/sodium citrate (SSC) at about 45xc2x0 C., followed by one or more washes in 0.2xc3x97 SSC, 0.1%SDS at 65xc2x0 C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5M Sodium Phosphate, 7% SDS at 65xc2x0 C., followed by one or more washes at 0.2xc3x97 SSC, 1% SDS at 65xc2x0 C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1,3, 5, 7, or 9, or corresponds to a naturally-occurring nucleic acid molecule.
As used herein, a xe2x80x9cnaturally-occurringxe2x80x9d nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
An xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language xe2x80x9csubstantially freexe2x80x9d means preparation of laminin 15 having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-laminin 15 protein (also referred to herein as a xe2x80x9ccontaminating proteinxe2x80x9d), or of chemical precursors or non-laminin 15 chemicals. When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.
A xe2x80x9cnon-essentialxe2x80x9d amino acid residue is a residue that can be altered from the wild-type sequence of a laminin 15 chain (e.g., the sequence of SEQ ID NO: 1, 3, 5, 7, or 9) without abolishing or more preferably, without substantially altering a biological activity, whereas an xe2x80x9cessentialxe2x80x9d amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present which mediate assembly and are predicted to be particularly unamenable to alteration.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a laminin 15 chain coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a laminin 15 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1, 3, 5, 7, or 9, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least and even 60%, more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid xe2x80x9cidentityxe2x80x9d is equivalent to amino acid or nucleic acid xe2x80x9chomologyxe2x80x9d). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J Mol Biol (48): 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a NWSgapdna. CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
xe2x80x9cMisexpression or aberrant expressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at nonwild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
A xe2x80x9cpurified preparation of cellsxe2x80x9d, as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.
The term xe2x80x9csubjectxe2x80x9d as used herein refers to a mammal. Examples of mammals include human and nonhuman primates, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, having a disorder associated with insufficient laminin, e.g., laminin 15 activity. The mammal is preferably a primate, e.g., a human.
As used herein the term xe2x80x9cadministeringxe2x80x9d refers to delivery of a preparation, composition, an active portion, or an active fragment of laminin 15 alone, in combination with another laminin (e.g., laminin 5, laminin 14) and/or at least one other compound or preparation.
The term xe2x80x9cstabilityxe2x80x9d means structural, anatomic molecular, and/or functional integrity, intactness, or completeness which is testable or observable by any suitable means. For example, the stability of retina photoreceptor can be tested by ERG, e.g., indicated by a wave and b wave.
The term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is intended to include a solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Such carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Pharmaceutically acceptable salts can also be used in the composition, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition can also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.
Liposomes, such as those described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 91114445; or EP 524,968 B1, can also be used as a carrier. Typically, the therapeutic laminin composition is prepared as an injectable, either as a liquid solution or suspension; however, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The composition can also be formulated into an entericcoated tablet or gel capsule according to known methods in the art, such as those described in U.S. Pat. No. 4,853,230; EP 225,189; AU 9,224,296; and AU 9,230,801.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.